Methods of enhancing functioning of the large intestine

ABSTRACT

The invention relates to glucagon-related peptides and their use for the prevention or treatment of disorders involving the large intestine. In particular, it has now been demonstrated that GLP-2 and peptidic agonists of GLP-2 can cause proliferation of the tissue of large intestine. Thus, the invention provides methods of proliferating the large intestine in a subject in need thereof. Further, the methods of the invention are useful to treat or prevent inflammatory conditions of the large intestine, including inflammatory bowel diseases.

This application is a continuation application of Ser. No. 10/419,150,filed Apr. 21, 2003, now allowed, which is a divisional application ofSer. No. 09/692,238, filed Oct. 20, 2000, now U.S. Pat. No. 6,589,399,which is a continuation of application Ser. No. 09/149,831, filed Sep.8, 1998, now U.S. Pat. No. 6,297,214, which is a continuation-in-part ofapplication Ser. No. 08/850,664, filed May 2, 1997, now abandoned.

FIELD OF INVENTION

This invention relates to glucagon-related peptides and their use forthe prevention or treatment of disorders involving the large intestine.

BACKGROUND OF THE INVENTION

Glucagon-like peptide-2 (GLP-2) is a 33 amino acid peptide expressed ina tissue-specific manner from the pleiotropic glucagon gene. GLP-2 showsremarkable homology in terms of amino acid sequence to glucagon andGlucagon-like peptide-1 (GLP-1). Further, different mammalian forms ofGLP-2 are highly conserved. For example, the human GLP-2 (hGLP-2) anddegu (a south American rodent) GLP-2 differ from rat GLP-2 (rGLP-2) byone and three amino acids respectively. When given exogenously, GLP-2can produce a marked increase in the proliferation of small intestinalepithelium of the test mice, apparently with no undesirable side effects(Drucker et al., 1996, PNAS:USA, 93:7911-7916). Subsequently it wasshown that peptide analogs of native GLP-2 with certain modifications tothe peptide sequence possess enhanced intestinotrophic activity at thesmall intestine (see co-pending application U.S. Ser. No. 08/669,791,incorporated herein by reference). Moreover, GLP-2 has also been shownto increase D-Glucose maximal transport rate across the intestinalbasolateral membrane [Cheeseman and Tseng, 1996, American Journal ofPhysiology 271:G477-G482].

SUMMARY OF THE INVENTION

The invention is based, in part, on the discovery that GLP-2 receptoragonists act to enhance functioning of the large intestine. It isaccordingly a general object of the present invention to exploit GLP-2receptor agonists for therapeutic and related purposes.

In particular, it has been demonstrated that GLP-2 and peptidic analogsof GLP-2 can cause proliferation of the tissue of large intestine. Thus,in one aspect the invention provides a method of proliferating the largeintestine in a subject in need thereof comprising delivering to thelarge intestine of the subject a large intestine proliferating amount ofGLP-2 or a GLP-2 analog. In a preferred embodiment, the GLP-2 analog isone that is resistant to cleavage by DPP-IV, e.g., human [Gly²]GLP-2(referred to herein as [Gly²]hGLP-2).

More particularly, and according to one aspect of the invention, thereis provided a method of treating a subject having an inflammatorycondition of the intestine involving the large intestine, wherein GLP-2or a GLP-2 analog is delivered to the large intestine in an amountcapable of ameliorating the inflammation of the large intestine. In apreferred embodiment, the GLP-2 analog is one that is resistant tocleavage by DPP-IV, e.g., [Gly²]hGLP-2.

In a related aspect of the invention, there is provided a method oftreating a subject having an inflamed large intestine comprising thestep of delivering to the subject a large intestine inflammationameliorating amount of GLP-2 or an analog of GLP-2 in a pharmaceuticallyor veterinarily acceptable carrier. In a further aspect, GLP-2 or ananalog of GLP-2 is provided in a pharmaceutically or veterinarilyacceptable form in an amount effective to cause proliferation of thelarge intestine. Preferably, the GLP-2 analog is one that is resistantto cleavage by DPP-IV, e.g., [Gly²]hGLP-2.

In another aspect, the invention provides a method of prophylacticallytreating a subject at risk of developing an inflammatory condition ofthe intestine involving the large intestine comprising the steps of

-   -   a) identifying a subject at risk of developing an inflammatory        bowel condition involving the large intestine; and    -   b) administering to the subject an amount of GLP-2 or a GLP-2        analog effective to inhibit onset of the inflammatory condition.

In another aspect of the invention, there is provided a method toidentify peptides useful to treat inflammatory conditions involving thelarge intestine comprising the steps of:

-   -   a) obtaining an analog of a vertebrate GLP-2 peptide, the analog        having at least one amino acid substitution, deletion, addition,        or an amino acid with a blocking group;    -   b) inducing an inflammatory condition of the intestine involving        the large intestine in a test animal;    -   c) treating the test animal having an induced inflammatory        condition of the large intestine, with the analog using a        regimen capable of eliciting an amelioration of the inflammatory        condition of the large intestine when utilized for human        [Gly²]GLP-2; and    -   d) determining the effect of the analog on the health status or        mortality of the test animal compared with control animals not        receiving the peptide or determining the mass of the large        intestine of test animals compared to control animals not        receiving peptide.

In a related aspect of the invention, there is provided a method usefulto identify peptides capable of proliferating the tissue of the largeintestine comprising the steps of:

-   -   a) obtaining an analog of a vertebrate GLP-2 peptide, the analog        having at least one amino acid substitution, deletion, addition,        or an amino acid with a blocking group;    -   b) delivering the analog to the large intestine of the test        animal using a regimen capable of proliferating the large        intestine when utilized for human [Gly²]GLP-2; and    -   c) assessing the increase in the mass or length of the large        intestine after completion of the treatment regime.

In another aspect, the invention provides a method for growing largeintestine tissue or cells therefrom, which comprises the step ofculturing the tissue or cells in a culturing medium supplemented with agrowth promoting amount of GLP-2 or a GLP-2 analog.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the change in total protein, and wet and dry weightof the large intestine after treatment with GLP-2. Female CD1 mice wereinjected with 2.5 μg of rat GLP-2 twice daily for 10 days. In FIG. 1A,segments of proximal jejunum (PJ), ileum (I), and colon (C) wereanalyzed for total protein concentration. FIG. 1B presents the wet anddry weight of 2 cm segments of proximal jejunum (PJ), ileum (I), andcolon (C).

FIG. 2 is a graph of the total body weight over time of mice withexperimentally induced colitis that were injected with PBS or with aGLP-2 agonist. The treatment groups are as indicated, and are furtherdescribed infra in Example 2. FIG. 2A—treatment groups 1 and 2. FIG.2B—treatment groups 3 and 4. FIG. 2C—treatment groups 5 and 6. FIG.2D—treatment groups 7 and 8.

FIG. 3 depicts the post-treatment large intestine mass in grams (g) ofeach treatment group described in Example 2 infra.

FIG. 4 graphs the post-treatment large intestine length in centimeters(cm) of each treatment group described in Example 2 infra.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to therapeutic and related uses of GLP-2 and GLP-2analogs, in particular for the amelioration of medical or veterinaryconditions in which functioning of the large intestine is impaired bydisease or injury. For example, the method is usefully applied to treatsubjects suffering from an inflammatory condition of the largeintestine, or subjects who have undergone resection of the largeintestine.

As used herein the terms “large intestine” means the distal portion ofthe intestine, extending from its junction with the small intestine tothe anus: it comprises the cecum, colon, rectum, and anal canal.

As used herein the term “subject” includes a human or other mammal andincluding livestock and pets.

As used herein the term “GLP-2 receptor agonist” means any moleculewhich on binding to the GLP-2 receptor results in activation of theGLP-2 receptor, and includes for example GLP-2 or peptidic analogs ofGLP-2. Recently it has been demonstrated that the GLP-2 receptor is aG-protein coupled receptor. Nucleic acid encoding the GLP-2 receptor hasbeen isolated [see co-pending applications U.S. Ser. No. 08/767,224,filed Dec. 13, 1996 and 08/845,546, filed Apr. 24, 1997, both of whichare incorporated herein by reference]. Thus, methods commonly used inthis field to identify G-protein coupled receptor agonists may beusefully applied to the GLP-2 receptor. One particularly usefulmethodology for assessing compounds for GLP-2 receptor agonist activityis disclosed in the above mentioned co-pending applications. Briefly,suitable cells such as COS cells are transformed with GLP-2 receptorencoding nucleic acid such that functional receptor is provided at thecell surface. Thereafter agonist activity of a test compounds can beassessed by contacting transformed cells by the test compound; anincrease in the intracellular level of cyclic adenosine monophosphate inresponse to binding of the test compound to the transformed cellsindicates agonist activity.

GLP-2 peptide analogs and selected chemical libraries, may be screenedfor GLP-2 receptor agonist activity using this approach. Guidance on thetypes of peptidic analogs that may be usefully employed in this methodis given herein and in co-pending application Ser. Nos. 08/632,533 and08/631,273, which are incorporated herein by reference. Moreover, any ofthe commercially available chemical libraries may be usefully screenedfor small molecule GLP-2 receptor agonists using high throughput orultra high throughput screening technology. Peptidic analogs of GLP-2and small molecule agonists identified as GLP-2 receptor agonists may bescreened for therapeutic and related utility to treat conditionsinvolving the large intestine using the models described herein.

Any subject requiring enhancement of the activity of the large intestinemay potentially be a candidate for treatment with a GLP-2 agonistaccording to the invention. In particular, one group of conditions thatmay be beneficially treated according to the invention are inflammatoryconditions of the intestine involving the large intestine (inflammatorybowel diseases, or “IBD”). Human patients are typically diagnosed ashaving such a condition after manifesting one or more of the followingsymptoms: pain in the abdomen, pain with defecation, diarrhea orconstipation (best described as a change in the normal bowel “habit”),rectal bleeding, fever, weight loss, anemia, fluid loss leading todehydration. Visualization using sigmoidoscopy or colonoscopy can beused to confirm the presence of an inflammatory condition of the largeintestine.

Alternatively, biopsies or a barium enema x-ray can be used to completethe diagnosis. Inflammatory bowel diseases include Crohn's disease andulcerative colitis, infectious colitis, drug or chemical-inducedcolitis, diverticulitis, and ischemic colitis. Standards for assessingthe severity of such diseases are well known in the art (see, forexample, Hanuer, 1996, New Eng. J. Med. 334:841-848).

Moreover, subjects identified to be at risk of developing an IBD andsubjects in remission from a condition involving inflammation of thelarge intestine may be beneficially treated prophylactically with aGLP-2 agonist according to the invention to inhibit onset ofinflammation of the large intestine. For example, ulcerative colitis andCrohn's disease can be familial diseases, accordingly, linkage studiescan identify suspectable individuals (see, for example, Hugot et al.,1996, Nature 379:821-823). Further, as it is known that the risk ofcolitis is increased in persons who have “quit” smoking, GLP-2 could beadvantageously administered to such subjects particularly suspectable ofdeveloping colitis.

Treatment with GLP-2 agonists has been demonstrated to increase thelength of the large intestine. Accordingly, subjects who would benefitfrom an increase in the length of the large intestine, for examplepatients who have undergone partial or non-total resection of the largeintestine, may be beneficially treated with GLP-2 receptor agonistsaccording to the invention.

A model suitable for determining which analogs of GLP-2 have largeintestine proliferation activity are potentially therapeutically usefulto treat medical or veterinary conditions of the large intestine isdescribed in Example 1.

Animal models useful for studying inflammatory conditions involving thelarge intestine are described in the literature. (See Elson et al.,1995, Gastroenterology 109:1344-1367; Kim et al., 1992, Scand. J.Gastroenterol. 27:529-537; Dieleman et al., 1994, Gastroenterology107:1643-1652; Domek et al., 1995, Scand. J. Gastroenterol.30:1089-1094; Mashimo et al., 1996, Science 274:262-265; Okayasu et al.,1990, Gastroenterology, 98:694-702; Takizawa et al., 1995, Adv. Exp.Med. Biol. 371:1383-1387; and Wells et al., 1990, J. Acquired Imm.Defic. Syndromes 3:361-365.) For example, ulcerative colitis isinducible in test mice using dextran sulphate (Okayasu et al., 1990,supra.) and is used herein in Example 2. Typically test mice ingesting3-10% dextran sulphate in their drinking water show at least one of thefollowing symptoms within 6-10 days: weight loss, rectal bleeding ordiarrhea, lethargy, weakness, and decreased movement, eating anddrinking. Thus, the animal model described in Example 2 can be used toassess the ability of compounds identified as GLP-2 agonist toameliorate inflammatory conditions involving the large intestine.

The various vertebrate forms of GLP-2 include, for example, rat GLP-2and its homologues including ox GLP-2, porcine GLP-2, degu GLP-2, bovineGLP-2, guinea pig GLP-2, hamster GLP-2, human GLP-2, rainbow troutGLP-2, and chicken GLP-2, the sequences of which have been reported bymany authors including Buhl et al. in J. Biol. Chem., 1988,263(18):8621, Nishi and Steiner, Mol. Endocrinol., 1990, 4:1192-8, andIrwin and Wong, Mol. Endocrinol., 1995, 9(3):267-77. The sequencesreported by these authors is incorporated herein by reference.

Analogs of vertebrate GLP-2 can be generated using standard techniquesof peptide chemistry and can be assessed for trophic activity at thelarge intestine, all according to the guidance provided herein.Particularly preferred analogs of the invention are those based upon thesequence of human GLP-2, as follows (SEQ ID NO: 1):

His-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp

wherein one or more amino acid residues are conservatively substitutedfor another amino acid residue, as long as the analog still maintainsits trophic activity at the large intestine as measured by an increasein at least one of the following parameters: large intestine length,protein content or mass.

Conservative substitutions in any naturally occurring GLP-2, preferablythe human GLP-2 sequence, are defined as exchanges within any of thefollowing five groups:

I. Ala, Ser, Thr, Pro, Gly II. Asn, Asp, Glu, Gln III. His, Arg, Lys IV.Met, Leu, Ile, Val, Cys V. Phe, Tyr, Trp.

The invention also encompasses non-conservative substitutions of aminoacids in any vertebrate GLP-2 sequence, provided that thenon-conservative substitutions occur at amino acid positions known tovary in GLP-2 isolated from different species. Non-conserved residuepositions are readily determined by aligning all known vertebrate GLP-2sequences. For example, Buhl et al., J. Biol. Chem., 1988, 263(18):8621,compared the sequences of human, porcine, rat, hamster, guinea pig, andbovine GLP-2′s, and found that positions 13, 16, 19, 27 and 28 werenon-conserved (position numbers refer to the analogous position in thehuman GLP-2 sequence). Nishi and Steiner, Mol. Endocrinol., 1990,4:1192-8, found that an additional position within the sequence encodingGLP-2, residue 20 in the above human sequence, also varied in degu, arodent species indigenous to South America. Thus, under this standard,the amino acid positions which vary in mammals and which preferable maybe substituted with non-conservative residues are positions 13, 16, 19,20, 27 and 28. The additional amino acid residues which vary invertebrates and which also may be substituted with non-conservedresidues occur at positions 2, 5, 7, 8, 9, 10, 12, 17, 21, 22, 23, 24,26, 29, 30, 31, 32 and 33.

Alternatively, non-conservative substitutions may be made at anyposition in which alanine-scanning mutagenesis reveals some tolerancefor mutation in that substitution of an amino acid residue with alaninedoes not destroy all intestinotrophic activity at the large intestine.The technique of alanine scanning mutagenesis is described by Cunninghamand Wells, Science, 1989, 244:1081, and incorporated herein by referencein its entirety. Since most GLP-2 sequences consist of onlyapproximately 33 amino acids (and in human GLP-2 alanine already occursat four positions), one of skill in the are could easily test an alanineanalogue at each remaining position for intestinotrophic effect, astaught in the examples below.

In specific embodiments of the invention, the GLP-2 peptide is selectedfrom

-   -   1) rat GLP-2 having the sequence illustrated below (SEQ ID NO:        2):

His-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-Ala-Thr-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp;

-   -   2) human GLP-2, the Thr¹⁹ to Ala¹⁹ equivalent of rat GLP-2,        illustrated below (SEQ ID NO: 1):

His-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp;

-   -   3) human [Gly²]GLP-2 (human GLP-2 wherein the alanine at        position 2 is replaced by a glycine);    -   4) GLP-2′s, and GLP-2 analogs, which incorporate an N-terminal        blocking group and/or an N-terminal extension such as Arg or        Arg-Arg; and/or incorporate a C-terminal blocking group and/or a        C-terminal extension such as Arg or Arg-Arg.

Guidance on particular analogs and variants of GLP-2 that may beusefully employed in the present invention, and guidance on how toproduce others, is provided in co-pending applications U.S. Ser. Nos.08/669,790 and 08/669,791, both filed on Jun. 28, 1996, the disclosuresof which are incorporated herein by reference. Briefly, anysubstitution, addition or deletion of GLP-2 that does not destroy theactivity of GLP-2 may be usefully employed in this invention. Inpreferred embodiments the GLP-2 analogs are at least as active as nativehuman GLP-2. In the most preferred embodiments, the GLP-2 analog hasenhanced activity compared with native human GLP-2. For example, suchanalogs may exhibit enhanced serum stability, enhanced receptor bindingand enhanced signal transducing activity. Other modifications to GLP-2and GLP-2 analogs that may usefully be employed in this invention arethose which render the molecule resistant to oxidation.

In a preferred embodiment of the invention, the GLP-2 analog is producedby the alteration of native GLP-2 to confer DPP-IV resistance, e.g., bysubstituting a Gly for Ala at position two. The DPP-IV-resistant classof GLP-2 analogs possess particularly advantageous properties. MammalianGLP-2 species have been found to be sensitive to cleavage by DPP-IVenzyme. This sensitivity to DPP-IV is the result of the recognitionsequence Ala² Asp³ found in all mammalian forms of GLP-2. In a preferredembodiment, the GLP-2 analogs are members of a class of GLP-2 analogswhich incorporate at position 2 and or position 3 a replacement aminoacid which confers on the GLP-2 analog relative resistance to DPP-IVmediated cleavage, as determined by any convenient in vitro or in vivoassessment technique that is able to detect the presence of GLP-2digestion products. A DPP-IV resistant GLP-2 analog is revealed as thatGLP-2 analog which is processed or degraded at a rate that is measurablyslower than the rate at which human GLP-2 is processed or degraded,under the same conditions.

The position two class of GLP-2 analogs is preferred herein. TheseAla²-substituted GLP-2 analogs can incorporate at position two astructurally wide variety of Ala-replacement amino acids to achieverelative resistance to DPP-IV digestion. A similarly wide variety ofAla-replacement amino acids allow also for the retention by the analogof intestinotrophic activity. For purposes of identifying thoseDPP-IV-resistant position two analogs that also retain intestinotrophicactivity, the position two analogs showing DPP-IV resistance arescreened in an assay of intestinotrophic activity. Such an assay isdescribed in co-pending application U.S. Ser. No. 08/669,791, filed onJun. 28, 1996, the disclosure of which is incorporated herein byreference.

In embodiments of the present invention, the Ala² replacements includestereoisomers of amino isomers that would otherwise be substrates forDPP-IV, for example D-Ala, D-HPr and D-Pro; naturally occurring aminoacids other than Ala, HPr and Pro which provide a basic or unchargedside chain, for example, Glu, Lys, Arg, Leu, Ile, Gly and Val.Particularly preferred GLP-2 analogs include [D-Ala²]rGLP-2,[Gly²]rGLP-2, [Val²]rGLP-2 and [Gly²]hGLP-2.

Further, a large number of agonist GLP-2 peptides that are described inPCT Application PCT/CA97/00252, filed Apr. 11, 1997, incorporated in itsentirety by reference herein, may also be used in the methods of theinvention.

The “blocking groups” represented by R1 and R2 are chemical groups thatare routinely used in the art of peptide chemistry to confer biochemicalstability and resistance to digestion by exopeptidase. SuitableN-terminal protecting groups include, for example, C₁₋₅alkanoyl groupssuch as acetyl. Also suitable as N-terminal protecting groups are aminoacid analogues lacking the amino function. Suitable C-terminalprotecting groups include groups which form ketones or amides at thecarbon atom of the C-terminal carboxyl, or groups which form esters atthe oxygen atom of the carboxyl. Ketone and ester-forming groups includealkyl groups, particularly branched or unbranched C₁₋₅ alkyl groups,e.g., methyl, ethyl, and propyl groups, while amide-forming groupsinclude amino functions such as primary amine, or alkylamino functions,e.g., mono-C₁₋₅alkylamino and di-C₁₋₅alkylamino groups such asmethylamino, ethylamino, dimethylamino, diethylamino, methylethylaminoand the like. Amino acid analogues are also suitable for protecting theC-terminal end of the present compounds, for example, decarboxylatedamino acid analogues such as agmatine.

The particular form of GLP-2 selected for promoting the growth of largeintestinal tissue can be prepared by a variety of techniques well knownfor generating peptide products. Vertebrate forms of GLP-2 can of coursebe obtained by extraction from the natural source, using an appropriatecombination of protein isolation techniques. As described by Buhl etal., supra, porcine GLP-2 isolation and purification is achieved fromacid-ethanol extracts of ileal mucosa by a combination of size selectionand HPLC-based fractionation, with the aid of antibody raised againstsynthetic proglucagon 126-159, to monitor work-up. As an alternative toGLP-2 extraction, those forms of GLP-2 that incorporate only L-aminoacids, whether vertebrate GLP-2 or analogs thereof, can be produced incommercial quantities by application of recombinant DNA technology. Forthis purpose, DNA coding for the desired GLP-2 or GLP-2 analog isincorporated into an expression vector and transformed into a microbial,e.g., yeast, or other cellular host, which is then cultured underconditions appropriate for GLP-2 expression. A variety of geneexpression systems have been adapted for this purpose, and typicallydrive expression of the desired gene from expression controls usednaturally by the chosen host. Because GLP-2 does not require posttranslational glycosylation for its activity, its production may mostconveniently be achieved in bacterial hosts such as E. coli.

For such production, DNA coding for the selected GLP-2 peptide mayusefully be placed under expression controls of the lac, trp or PL genesof E. coli. As an alternative to expression of DNA coding for the GLP-2per se, the host can be adapted to express GLP-2 peptide as a fusionprotein in which the GLP-2 is linked releasable to a carrier proteinthat facilitates isolation and stability of the expression product.

In an approach universally applicable to the production of a selectedGLP-2 or GLP-2 analog, and one used necessarily to produce GLP-2peptides that incorporate non-genetically encoded amino acids and N- andC-terminally derivatized forms, the well established techniques ofautomated peptide synthesis are employed, general descriptions of whichappear, for example, in J. M. Stewart and J. D. Young, Solid PhasePeptide Synthesis, 2nd Edition, 1984 Pierce Chemical Company, Rockford,Ill.; and in M. Bodanszky and A. Bodanszky, The Practice of PeptideSynthesis, 1984, Springer-Verlag, New York; Applied Biosystems 430AUsers Manual, 1987, ABI Inc. Foster City, Calif. In these techniques,GLP-2 peptide is grown from its C-terminal, resin-conjugated residue bythe sequential addition of appropriately protected amino acids, usingeither the Fmoc or tBoc protocols, as described for instance by Orskovet al., Febs Letters, 1989, 247(2):193-196.

For the incorporation of N- and/or C-blocking groups, protocolsconventional to solid phase peptide synthesis methods can also beapplied. For incorporation of C-terminal blocking groups, for example,synthesis of the desired peptide is typically performed using, as solidphase, a supporting resin that has been chemically modified so thatcleavage from the resin results in a GLP-2 peptide having the desiredC-terminal blocking group. To provide peptides in which the C-terminusbears a primary amino blocking group, for instance, synthesis isperformed using a p-methylbenzhydrylamine (MBHA) resin so that, whenpeptide synthesis is completed, treatment with hydrofluoric acidreleases the desired C-terminally amidated peptide. Similarly,incorporation of an N-methylamine blocking group at the C-terminus isachieved using N-methylaminoethyl-derivatized DVB resin, which upon HFtreatment releases peptide bearing an N-methylamidated C-terminus.Protection of the C-terminus by esterification can also be achievedusing conventional procedures. This entails use of resin/blocking groupcombination that permits release of side-chain protected peptide fromthe resin, to allow for subsequent reaction with the desired alcohol, toform the ester function. FMOC protecting groups, in combination with DVBresin derivatized with methoxyalkoxybenxyl alcohol or equivalent linker,can be used for this purpose, with cleavage from the support beingeffected by TFA in dichloromethane. Esterification of the suitablyactivated carboxyl function, e.g., with DCC, can then proceed byaddition of the desired alcohol, followed by deprotection and isolationof the esterified GLP-2 peptide.

Incorporation of N-terminal blocking groups can be achieved while thesynthesized GLP-2 peptide is still attached to the resin, for instanceby treatment with suitable anhydride and nitrile. To incorporate anacetyl blocking group at the N-terminus, for instance, the resin-coupledpeptide can be treated with 20% acetic anhydride in acetonitrile. TheN-blocked GLP-2 peptide can then be cleaved from the resin, deprotectedand subsequently isolated.

Once the desired GLP-2 peptide has been synthesized, cleaved from theresin and fully deprotected, the peptide is then purified to ensure therecovery of a single oligopeptide having the selected amino acidsequence.

Purification can be achieved using any of the standard approaches, whichinclude reversed-phase high-pressure liquid chromatography (RP-HPLC) onalkylated silica columns, e.g., C₄-, C₈-, or C₁₈-silica. Such columnfractionation is generally accomplished by running linear gradients,e.g., 10-90%, of increasing % organic solvent, e.g., acetonitrile, inaqueous buffer, usually containing a small amount (e.g., 0.1%) ofpairing agent such as TFA or TEA. Alternatively, ion-exchange HPLC canbe employed to separate peptide species on the basis of their chargecharacteristics. Column fractions are collected, and those containingpeptide of the desired/required purity are optionally pooled. In oneembodiment of the invention, the GLP-2 peptide is then treated in theestablished manner to exchange the cleavage acid (e.g., TFA) with apharmaceutically acceptable acid, such as acetic, hydrochloric,phosphoric, maleic, tartaric, succinic and the like, to generate apharmaceutically acceptable acid addition salt of the peptide.

For administration to patients, the GLP-2 peptide or its salt isprovided, in one aspect of the invention, in pharmaceutically acceptableform, e.g., as a preparation that is sterile-filtered, e.g., through a0.22μ filter, and substantially pyrogen-free. Desirably, the GLP-2peptide to be formulated migrates as a single or individualized peak onHPLC, exhibits uniform and authentic amino acid composition and sequenceupon analysis thereof, and otherwise meets standards set by the variousnational bodies which regulate quality of pharmaceutical products.

For therapeutic use, the chosen GLP-2 or GLP-2 analog is formulated witha carrier that is pharmaceutically acceptable and is appropriate foradministering the peptide to the subject by the chosen route ofadministration so as to deliver the peptide to the large intestine.Suitable pharmaceutically acceptable carriers are those usedconventionally with peptide-based drugs, such as diluents, excipientsand the like. Reference may be made to “Remington's PharmaceuticalSciences”, 17th Ed., Mack Publishing Company, Easton, Pa., 1985, forguidance on drug formulations generally. In one embodiment of theinvention, the compounds are formulated for administration by infusion,e.g., when used as liquid nutritional supplements for patients on totalparenteral nutrition therapy, or by injection, e.g., sub-cutaneously,intramuscularly or intravenously, and are accordingly utilized asaqueous solutions in sterile and pyrogen-free form and optionallybuffered to physiologically tolerable pH, e.g., a slightly acidic orphysiological pH. Thus, the compounds may be administered in a vehiclesuch as distilled water or, more desirably, in saline, phosphatebuffered saline or 5% dextrose solution. Water solubility of the GLP-2or GLP-2 analog may be enhanced, if desired, by incorporating asolubility enhancer, such as acetic acid or sodium hydroxide.

The aqueous carrier or vehicle can be supplemented for use asinjectables with an amount of gelatin effective to achieve the depoteffect are expected to lie in the range from 10-20%. Alternative gellingagents, such as hyaluronic acid, may also be useful as depoting agents(also veterinary applications).

As an alternative to injectable formulations, the GLP-2 or GLP-2 analogmay be formulated for administration to patients and delivery to thelarge intestine by other routes.

Oral dosage forms, such as tablets, capsules and the like, can beformulated in accordance with standard pharmaceutical practice.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

The GLP-2′s and GLP-2 analogs of the invention may also be formulated asa slow release implantation device for extended and sustainedadministration of GLP-2. Examples of such sustained release formulationsinclude composites of bio-compatible polymers, such as poly(lacticacid), poly(lactic-co-glycolic acid), methylcellulose, hyaluronic acid,collagen, and the like. The structure, selection and use of degradablepolymers in drug delivery vehicles have been reviewed in severalpublications, including, A. Domb et al., Polymers for AdvancedTechnologies 3:279-292 (1992). Additional guidance in selecting andusing polymers in pharmaceutical formulations can be found in the textby M. Chasin and R. Langer (eds.), “Biodegradable Polymers as DrugDelivery Systems”, Vol. 45 of “Drugs and the Pharmaceutical Sciences”,M. Dekker, New York, 1990. Liposomes may also be used to provide for thesustained release of a GLP-2 or GLP-2 analog. Details concerning how touse and make liposomal formulations of drugs of interest can be foundin, among other places, U.S. Pat. No. 4,921,706; U.S. Pat. No.5,008,050; U.S. Pat. No. 4,921,706; U.S. Pat. No. 4,927,637; U.S. Pat.No. 4,452,747; U.S. Pat. No. 4,016,100; U.S. Pat. No. 4,311,712; U.S.Pat. No. 4,370,349; U.S. Pat. No. 4,372,949; U.S. Pat. No. 4,529,561;U.S. Pat. No. 5,009,956; U.S. Pat. No. 4,725,442; U.S. Pat. No.4,737,323; U.S. Pat. No. 4,920,016. Sustained release formulations areof particular interest when it is desirable to provide a high localconcentration of a GLP-2 or GLP-2 analog, e.g., near or at the largeintestine to promote large intestine growth in colitis etc.

The GLP-2′s and GLP-2 analogs of the invention may also be administeredto a subject in admixture with at least one other peptide hormoneselected from the group consisting of IGF-1, IGF-2 and GH. Suchcombinations have been shown to have synergistic effects on the growthof the large bowel (see U.S. patent application Ser. No. 08/763,177,filed Dec. 10, 1996, incorporated herein by reference). However, as ithas been found that GLP-2 alone can stimulate large intestine growth andameliorate inflammatory conditions of the large bowel, the inventionencompasses GLP-2 and GLP-2 analog formulations which do not containIGF-1, IGF-2 or GH peptide hormones.

For use in stimulating growth of the large intestine in a mammalincluding a human, the present invention provides in one of its aspectsa package, in the form of a sterile-filled vial or ampoule, thatcontains a tissue growth promoting amount of the GLP-2 or GLP-2 analog,in either unit dose or multi-dose amounts, wherein the packageincorporates a label instructing use of its contents for the promotionof such growth. In one embodiment of the invention, the package containsthe GLP-2 or GLP-2 analog and the desired carrier, as anadministration-ready formulation.

Alternatively, and according to another embodiment of the invention, thepackage provides the GLP-2 or GLP-2 analog in a form, such as alyophilized form, suitable for reconstitution in a suitable carrier,such as phosphate-buffered saline.

In one embodiment, the package is a sterile-filled vial or ampoulecontaining an injectable solution which comprises an effective, largeintestine proliferating amount of GLP-2 or GLP-2 analog dissolved in anaqueous vehicle.

According to the present invention, the GLP-2 or GLP-2 analog isadministered to treat patients that would benefit from growth of thetissue of the large intestine. In general, patients who would benefitfrom either increased large intestinal mass and consequent increasedlarge intestine mucosal function are candidates for treatment with GLP-2or GLP-2 analog. Particular conditions that may be treated with GLP-2include the various forms of inflammatory bowel disease includingcolitis and IBD, as well as patients who have undergone partial orsub-total resection of the large intestine. The therapeutic efficacy ofthe GLP-2 treatment may be monitored by: subjective improvement inabdominal pain, diarrhea or rectal bleeding; weight gain; normalizationof hemoglobin or white blood cell count and sedimentation rate; improvedappearance of the intestine on colonoscopy or sigmoidoscopy, improvementof intestine function as assessed radiologically by barium enema;histological improvement as assessed by specimen biopsy; and byamelioration (reduction or elimination) of the symptoms associated withthese conditions. For example, GLP-2 or GLP-2 analog is administrated toa patient with an inflammatory condition involving the large intestinein an amount sufficient to ameliorate the intestinal discomfort,bleeding and diarrhea caused by the condition. Additionally, GLP-2 orGLP-2 analog may be administered to patients who are identified as beingat risk of developing IBD.

The therapeutic dosing and regimen most appropriate for patienttreatment will of course vary with the disease or condition to betreated, and according to the patient's weight and other parameters. Theresults presented hereinbelow demonstrate that a dose of GLP-2 or GLP-2analog equivalent to about 1 mg/kg (or less, see below) administeredtwice daily over 10 days can ameliorate inflammatory conditions of thelarge intestine. It is expected that much smaller doses, e.g., in theμg/kg range, and shorter or longer duration or frequency of treatment,will also produce therapeutically useful results, i.e., a statisticallysignificant increase particularly in large intestine mass. The dosagesizes and dosing regimen most appropriate for human use are guided bythe results herein presented, and can be confirmed in properly designedclinical trials.

An effective dosage and treatment protocol may be determined byconventional means, starting with a low dose in laboratory animals andthen increasing the dosage while monitoring the effects, andsystematically varying the dosage regimen as well. Numerous factors maybe taken into consideration by a clinician when determining an optimaldosage for a given subject. Primary among these is the amount of GLP-2normally circulating in the plasma, which is on the order of 151 pmol/mLin the resting state, rising to 225 pmol/mL after nutrient ingestion forhealthy adult humans (Orskov, C. and Holst, J. J., 1987, Scand. J. Clin.Lab. Invest. 47:165). Additional factors include the size of thepatient, the age of the patient, the general condition of the patient,the particular disease being treated, the severity of the disease, thepresence of other drugs in the patient, the in vivo activity of theGLP-2 peptide and the like. The trial dosages would be chosen afterconsideration of the results of animal studies and the clinicalliterature. It will be appreciated by the person of ordinary skill inthe art that information such as binding constants and Ki derived fromin vitro GLP-2 binding competition assays may also be used incalculating dosages.

A typical human dose of a GLP-2 peptide would be from about 10 μg/kgbody weight/day to about 10 mg/kg/day, preferably from about 50μg/kg/day to about 5 mg/kg/day, and most preferably about 100 μg/kg/dayto 1 mg/kg/day.

In another of its aspects, the invention provides for the treatment ofpatient candidates as just identified using implanted cells that haveeither been conditioned in vitro or in vivo by prior incubation ortreatment with GLP-2 or GLP-2 analog, or have been engineeredgenetically to produce it. Conditioning of the cells ex vivo can beachieved simply by growing the cells or tissue to be transplanted in amedium that has been supplemented with a growth-promoting amount of theGLP-2 or GLP-2 analog and is otherwise appropriate for culturing ofthose cells. The cells can, after an appropriate conditioning period,then be implanted either directly into the patient or can beencapsulated using established cell encapsulation technology, and thenimplanted.

Yet another aspect of the invention encompasses treating animals in vivowith GLP-2 peptides in order to promote the growth of large intestinetissue. After subsequent enlargement of the large intestine thesetissues may then be used in a xenotransplantation procedure. Such GLP-2peptide treatment can be advantageous prior to xenotransplantation ofthe tissue from a non-human animal to a human because the size of thetransplanted organ or tissue often limits the success of this procedure.For example, a porcine donor animal may be treated with GLP-2 peptide inorder to increase large intestine size prior to xenotransplantation ofthe porcine large intestine tissue into a human in need of this organ.

Alternatively, the cells to be implanted can be raised in vitro from acell that has been engineered genetically to express or to over-expresseither the glucagon gene or, more directly, DNA coding solely for GLP-2.The sequence of such DNA can readily be determined from the amino acidsequence of the selected GLP-2, with the limitation that only GLP-2forms containing genetically encoded amino acids can be produced in thismanner. Various viral vectors, suitable for introduction of geneticinformation into human cells, can be employed and will incorporate theGLP-2-encoding DNA under expression controls functional in the hostcells. Once altered genetically, the engineered cells can then beimplanted using procedures established in the art. (See, for example,Drucker et al., 1996, PNAS:USA, 93:7911-7916.)

The invention having been described, the following examples are offeredby way of illustration and not limitation.

Example 1

In this experiment, the effect of GLP-2 on both small and largeintestinal mass, as well as the relative contribution of water to theincrement in intestine weight, was examined.

Female CD1 mice (6 weeks old) were treated with rat GLP-2, 2.5 μg twicedaily (subcutaneously) for 10 days. Following treatment, mice werefasted 24 hours, anesthetized with CO₂ and sacrificed. The small andlarge intestine was removed from the peritoneal cavity (from pylorus tocecum), cleaned, weighed and measured. For comparative purposes, 2 cmsegments of proximal jejunum, ileum and colon were obtained from eachanimal from the identical anatomical positions (as measured from thepylorus and cecum) and analyzed for protein content, wet weight and dryweight (expressed as mean±S.E.M in mg/2 cm segment).

For protein content, two 2 cm segments from the proximal jejunum, ileumand colon were removed, placed in 13 ml tubes containing 2 ml of PBS,homogenized for 20 seconds in a Brinkmann Homogenizer and placed on ice.The tubes were centrifuged for 5 min. at 1000 g and 100 μl aliquots ofsupernatant homogenate removed for determination of protein contentusing the modified Bradford method.

To measure wet and dry weight, 2 cm intestinal segments were weighed,placed in culture tubes, reweighed, then freeze-dried overnight in aFast-Freeze flask (Labconco) and reweighed.

Results are shown in FIGS. 1A and 1B. Both the wet and dry weights ofintestinal segments from the proximal jejunum were significantlyincreased following treatment with GLP-2 (p<0.05). The weights of thesegments obtained from the colon were slightly, but consistently,increased after GLP-2 treatment. A statistically significant increase inprotein content was observed in both the jejunum and colon ofGLP-2-treated mice as compared to PBS-treated mice.

Example 2

This experiment was designed to test the activity of GLP-2 inameliorating disease symptoms in an animal model of colitis. A 5%dextran sulfate solution was used to induce ulcerative colitis in testmice (Okayasu et al., 1990, 98:694-702).

Forty female CD1 mice (six-weeks old and approximately 25 g, obtainedfrom Charles River) were used in the experiment. On day 1, the mice wereweighed using a Mettler PJ300 scale and randomly allocated to 1 of eighttreatment groups. The groups, each consisting of 5 mice housed together,were treated as follows:

Groups 1 and 2—Controls

These two groups were fed normal drinking water.

Group 1) Ten days of injections (PBS), day 7 to day 17, animalssacrificed day 17.

Group 2) Ten days of injections human (Gly 2) GLP-2 (2.5 μg bid), day 7to day 17, animals sacrificed day 17.

Groups 3 and 4—Test Group

Drinking water given to these groups contained 5% Dextran sulfate (DS).

Group 3) DS given from day 7 to day 17, PBS injections given from day 7to day 17, animals sacrificed day 17.

Group 4) DS given from day 7 to day 17, human[Gly²] GLP-2 (2.5 μg bid)injections given day 7 to day 17, animals sacrificed day 17.

Groups 5 and 6—Test Group

Drinking water contained 5% Dextran sulfate day 2 to day 9, followed by4 days of normal drinking water. Ten days of injections were started day4.

Group 5) DS from day 2 to day 9, switch to normal water day 10 to day14, injections (PBS) from day 4 to day 14, animals sacrificed day 14.

Group 6) DS from day 2 to day 9, switch to normal water day 10 to day14, human [Gly²]GLP-2 (2.5 μg bid) injections given day 4 to day 14,animals sacrificed day 14.

Groups 7 and 8—Test Group

Animals were given eight days of drinking water containing 5% Dextransulfate, followed by 4 days normal water. Ten days of injections started4 days after the start of the 5% Dextran sulfate regimen.

Group 7) DS from day 2 to day 9, switch to normal water day 10, PBSinjections given from day 6 to day 16, animals sacrificed day 16.

Group 8) DS from day 2 to day 9, switch to normal water day 10, human[Gly²]GLP-2 (2.5 μg bid) day 6 to day 16, animals sacrificed day 16.

Dextran sulfate (USB, Cleveland, Ohio, MW 40,000-50,000) was dissolvedinto double distilled water, and placed into the water bottles. Waterbottles were topped up daily.

GLP-2 peptide was reconstituted and aliquoted on the day prior to thefirst day of injection and kept in a −20 C freezer. Human [Gly²]GLP-2 (5mg/mL) was reconstituted in PBS. One microliter of 5N NaOH was requiredto dissolve the peptide in a volume of 1 ml. The injection volume of 0.5ml was constant throughout the experiment using ½ cc U-100 InsulinSyringes Becton Dickinson and Company, NJ). Control mice (cage 1)received the same volume (0.5 ml) of phosphate buffered saline (PBS—137mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄.7H₂O, 1.4 mM KH₂PO₄, pH 7.3).

Subcutaneous injections in the right hind quarter began on various daysdepending on the treatment conditions given above. All injections weretwice a day, at 8 am and 6 pm. Mice in cages 1 and 2 were fasted on theday prior to sacrifice. Body weight measurements were made on day 1, day7, day 12 and the day of sacrifice. The weight and length of the largeintestine of each animal was measured after sacrifice.

Average body weights for each group during the course of the experimentare presented graphically in FIG. 2. The results for groups 3 and 4demonstrate that test animals given injections of an analog of GLP-2maintain body weight better through the course of the experiment andhave significantly heavier large intestines at sacrifice than miceinjected with PBS.

FIG. 3 illustrates the effects of treatment on post-treatment largeintestinal mass. As described in Example 1, administration of GLP-2agonist causes an increase in large intestinal mass (compare groups 1and 2). Similarly, when GLP-2 agonist is provided at the same time asinduction of colitis by dextran sulfate, GLP-2 agonist ameliorates thesevere morbidity, dehydration, and reduction in mass of the largeintestine caused by extended periods of induced colitis (compare groups3 and 4). However, shorter periods of chemically induced colitis causeda generalized inflammation and consequent increase in weight of thelarge bowel (groups 5 and 7).

GLP-2 agonist also caused a significant increase in large intestinelength, as shown in FIG. 4 (compare groups 1 and 2). This effect wasalso exhibited, although to a lesser degree, during experimentallyinduced colitis (compare groups 3 and 4).

Thus, the above results demonstrate that GLP-2 agonist not only has anameliorating effect on the symptoms of inflammatory conditions involvingthe large intestine (when provided at the same time as intestineinflammation is induced), but also causes an increase in both largeintestinal weight and length.

Example 3

In the following experiment, GLP-2 agonist was shown to decreasemortality in mice exposed to high levels of colitis-inducing dextransulphate.

Two groups of mice, 5-6 weeks of age, (10 mice per group) were given adlibitum access to drinking water supplemented with 10% dextran sulfate(same protocol as in Example 2, only increased concentration of dextransulfate). Mice were also treated with 2 subcutaneous injections per day(0.5 ml) of either saline, or [GLY²]hGLP-2, 2.5 μg per injection. After9 days, the experiment was stopped.

Only 3 of 10 mice in the saline-treated group survived, whereas 7 of 10mice in the GLP-2 agonist-treated group survived. Accordingly,GLP-2-treated mice were better resistant to the effects of dextransulphate, and had decreased mortality, than mice treated with PBS alone.

EQUIVALENTS

The foregoing written specification is sufficient to enable one skilledin the art to practice the invention. Indeed, various modifications ofthe above-described means for carrying out the invention which areobvious to those skilled in the field of molecular biology, medicine orrelated fields are intended to be within the scope of the followingclaims.

1. A method for treating a subject to enhance functioning of the largeintestine, comprising the step of delivering to the large intestine ofthe subject a GLP-2 receptor agonist in an amount effective to enhancefunctioning of the large intestine.
 2. A method of treating a subject toproliferate the tissue of the large intestine, comprising the step ofdelivering to the large intestine of the subject GLP-2 or a peptidicanalog of GLP-2, in an amount effective to proliferate the tissue of thelarge intestine.
 3. A method according to claim 2, wherein the subjectis suffering from an inflammatory condition involving the largeintestine.
 4. A method according to claim 3, wherein the inflammatorycondition involving the large intestine is selected from the groupcomprising Crohn's disease, ulcerative colitis, infectious colitis, drugor chemical-induced colitis, diverticulitis, and ischemic colitis.
 5. Amethod according to claim 2, wherein the subject has undergone partialor subtotal resection of the large intestine.
 6. A method according toclaim 2, wherein the subject is a human.
 7. A method according to claim6, wherein the analog of GLP-2 has enhanced large intestine cellproliferating activity relative to native rat GLP-2.
 8. A methodaccording to claim 7, wherein the analog of GLP-2 is resistant tocleavage by DPP-IV.
 9. A method according to claim 8, wherein the analogof GLP-2 is selected from the group consisting of [D-Ala²]rGLP-2,[Gly²]rGLP-2, [Val²]rGLP-2, and [Gly²]hGLP-2.
 10. A method according toclaim 8, wherein the analog of GLP-2 is [Gly²]hGLP-2.
 11. A methodaccording to claim 8, wherein the analog of GLP-2 is delivered to thelarge intestine by oral, subcutaneous, or rectal administration.
 12. Amethod to identify peptides useful to treat inflammatory conditionsinvolving the large intestine, comprising the steps of: a) obtaining ananalog of a vertebrate GLP-2 peptide, the analog having at least oneamino acid substitution, deletion, addition, or an amino acid with ablocking group; b) inducing an inflammatory condition of the intestineinvolving the large intestine in a test animal; c) treating the testanimal having an induced inflammatory condition of the large intestine,with the analog using a regimen capable of eliciting an amelioration ofthe inflammatory condition of the large intestine when utilized fornative GLP-2; and d) determining the effect of the analog on the healthstatus or mortality of the test animal compared with control animals notreceiving the peptide or determining the effect of the analog on theweight of the large intestine of test animals compared to controlanimals not receiving peptide.
 13. A method useful to identify peptidescapable of proliferating tissue of the large intestine, comprising thesteps of a) obtaining an analog of a vertebrate GLP-2 peptide, theanalog having at least one amino acid substitution, deletion, addition,or an amino acid with a blocking group; b) delivering the analog to thelarge intestine of the test animal using a regimen capable of elicitingproliferation of the large intestine when utilized for native GLP-2; andc) assessing the increase in the mass or length of the large intestineafter completion of the treatment regimen.
 14. A method ofprophylactically treating a subject at risk of developing aninflammatory condition of the intestine involving inflammation of thelarge intestine, the method comprising: a) identifying a subject at riskof developing an inflammatory bowel condition involving the largeintestine; b) administering to the subject an amount of GLP-2 or a GLP-2analog demonstrates to inhibit onset of the inflammatory condition.